Highly pathogenic avian influenza virus H5N1 poses a debilitating pandemic threat. While prophylactic vaccines are being developed, ongoing viral evolution to evade immune responses remains problematic. Therefore, my long-term goals are to understand the antigenic evolution and molecular epidemiology of this virus in the face of selective immune pressure and to gain insights into the genesis and emergence of potentially pandemic strains. The central hypothesis is that viral escape mutants are constrained by specific interactions with neutralizing antibodies, and these mutants must evolve along defined, dominant, and reproducible evolutionary routes to escape from the immune pressure. The hypothesis is based, in part, on observations from epidemiological analysis of H5N1 virus from recent outbreaks in human populations. Some amino acids changes in hemagglutinin (HA) were involved in receptor binding and more frequently observed in human than in avian isolates, indicating that these viruses were antigenically distinct. This positive selection may be due to specific immune pressures as well as the process of viral adaptation to human hosts. I will test the hypothesis using human mAbs against the HA to define the pathways of H5N1 virus evolution under selective immune pressures. Additionally, I will correlate the specific routes of escape with clinical outcomes of influenza infection. In particular, I propose the following specific aims: 1) To characterize the epitopes of a panel of neutralizing and cross-neutralizing anti-H5 antibodies and their mechanisms of action. 2) To identify viral escape mutants induced by these antibodies and delineate the permitted evolutionary routes, as defined by patterns of sequence variations, by which the virus can escape epitope-specific antibody neutralization. 3) To determine if routes of virus evolution can be correlated with viral fitness and pathogenicity in vitro and clinical outcome of viral infection in vivo. RELEVANCE: Preventing human infections with bird flu has become a major public health priority as the virus continues to cause widespread economic and human losses as it spreads. The scientific contributions of this research will add to our understanding of how the virus survives detection and elimination by the immune system and provide strategic approaches to preventing infection or clinical disease progression.